In article <4oet96$ch5 at sunca.ncaur.gov>, skorycd at ncaur1.ncaur.gov says...
>>I have always made my PCR and sequencing primers with a G or C
>at the 3' end to presumably stabilize it during extension. While I am
>not aware of any research papers to support this logic, I have seen
>several manufactures (eg: PerkinElmer) recommend a similar
>design.
>
Dear Chris,
it makes sense, but in most cases you won't notice the difference. If your
gene is very rich in GC's a 3'end like x(N)ggCCg for example may lead to
unspecific priming just with this end. The reason to avoid stretches is the
fact, that there is some bias towards long stretches of 5 or more
identical nucleotides. This is worst for G/C, besides the A/T's resulting from
polyadenylation sites in cDNA libraries. There was some paper about this many
years ago in JMB(?). As the polymerase will remove the first 3' base when
starting polymerisation, it may not be enough to add only one base beyond such
stretches in your primer to maintain specifity.
5' does certainly not matter, but your oligo should be stable still. I found
Oligo4.0 very helpful in predicting the TM, especially to avoid too weak or
too stable primers (TM>>60 deg C). The secondary structure options of these
programmes are certainly helpful, but I agree with the all the other RE:, that
by eye inspection very often is enough.
G.L. Michael
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